The invention relates to the production of RNA free cellular components, and a method of removing RNA from preparations of cellular components.
The ribonuclease enzyme family (hereafter referred to as RNases) has been extensively studied. Numerous RNases have been characterized and the genes encoding some of these proteins have been cloned.
RNases hydrolyze one or more of the phosphodiester bonds in single stranded and double stranded RNA as well as RNA in RNA:DNA hybrids. RNases differ in their specificity for either a particular form of the RNA substrate (for example single stranded, double stranded or in a DNA:RNA hybrid) or their specific point of RNA cleavage.
One biological activity of RNase enzymes is the processing of mature molecules of RNA from precursor forms (Genes, Benjamin Lewin ed., John Wiley and Sons, 2nd edition, p.395, 1985). Certain RNases can affect the growth and differentiation of mammalian cells by virtue of an intrinsic anti-tumor activity (references within Ribo et al., Prot. Express. and Purif. 7: 253-261, 1996).
RNA is a major contaminant of preparations from cell lysates. For example, plasmid DNA preparations contain RNA that is difficult to remove by anion exchange or size exclusion chromatography because it is similar in size (with respect to exclusion by SEC matrices) and charge to DNA. RNase A hydrolyzes RNA after C and U residues by cleaving between the 3xe2x80x2-phosphate group of a pyrimidine ribonucleotide and the 5xe2x80x2-hydroxyl of the adjacent nucleotide. This enzyme is used to degrade RNA to low molecular weight species that no longer copurify with plasmid or genomic DNA. RNase I can also be used to remove RNA from preparations of plasmid or genomic DNA. RNase A is also commonly used for the enzymatic digestion of host derived RNA during the production of recombinant protein from E. coli. 
A prior art method of removing RNA from a sample is to add a large quantity of an exogenously produced RNase. For example, to remove RNA from plasmid DNA bovine RNase A is added to a final concentration of 100 xcexcg/ml (QIAGEN Plasmid Handbook February 1995, QIAGEN Ltd. Unit 1 Tillingbourne Court, Dorking Business Park, Dorking, Surrey RH4 1HJ, UK). Bovine RNase A is commonly used at a final concentration of approximately 10-100 xcexcg/ml for the enzymatic digestion of host derived RNA during the production of recombinant protein from E. coli. A chief disadvantage of prior art methods is that the cellular component that is treated according to these methods often contains residual RNase of animal origin.
There are limitations to using exogenously produced RNases. First, it is difficult to purify large amounts of RNase from the tissue of origin. This is presumably because high concentrations of active RNase will degrade host cell RNA and impair normal cell functions to a level that can be toxic to a cell. Therefore, it is difficult to produce large quantities of active RNase by expression in cells because high concentrations of active RNase will be toxic to a cell. It is also difficult to produce large quantities of active RNase by expression in cells because RNase can be sensitive to proteases of the host in which it is being synthesized, and because RNases that are over-produced and accumulate as in e.g. E. coli inclusion bodies cannot always be correctly refolded. The most significant limitation to using exogenously produced RNase is that, if the exogenously added RNase is of animal origin, following RNase treatment the presence of residual enzyme can contaminate a DNA preparation, thereby rendering it unacceptable for certain applications, including gene therapy. Third, it is expensive to produce RNase in large quantities.
There is a need in the art for plasmid DNA and protein preparations that are substantially free from contaminating RNA.
There is a need in the art for methods of producing RNA-free cellular components that are suitable for administration to human subjects.
There is a need in the art for a method of removing RNA from cellular components that does not rely on incubating a cellular lysate or purified component with added exogenously produced RNase.
The invention features a method of preparing a substantially RNA-free cellular component comprising culturing cells producing the cellular component in a medium and lysing said cells to produce a cell lysate, wherein said cell lysate contains said cellular component and sufficient RNase activity to degrade substantially all of the RNA molecules present in said cell lysate.
In a preferred embodiment, the RNase is produced by cells producing the cellular component.
Alternatively, the RNase is produced by cells in the medium other than those cells producing the cellular component.
The invention also features a method of preparing a substantially RNA-free cellular component, comprising culturing and lysing cells producing the cellular component and cells producing an RNase in an amount sufficient to degrade substantially all of the RNA present in the preparation.
Preferably, the cells producing the cellular component also produce the RNase and the culture and lysate contain cells producing the cellular component and an RNase in an amount sufficient to degrade substantially all of the RNA present in the preparation. Preferably, the cellular component and the RNase are produced by the same cell.
In preferred embodiments of both of the above-described inventive methods, the cellular component is one of a DNA, a protein, and a carbohydrate. Preferably, the cellular component is one of a recombinant DNA, a recombinant protein and a recombinant carbohydrate. Preferably, the RNase is encoded on a plasmid and the cellular component is encoded on the same plasmid, another plasmid or on the cell""s chromosome.
In preferred embodiments of both of the above-described inventive methods the gene encoding said RNase is integrated into the genome of the cell producing the RNase.
In some methods of the invention, it is preferred that the RNase is non-specific. Such a non-specific RNase may be RNase A, RNase M of RNase I.
In preferred embodiments of both of the above-described inventive methods, a cell producing an RNase produces the RNase in a regulated manner.
Preferably, the RNase produced by the host cell is overproduced, either by inducible production, or by constitutive production. The RNase overproduced by the host cell also may be secreted out of the host cell cytoplasm, for example, secreted into the host cell periplasm or secreted out of the host cell into the medium.
In some methods, it is preferred that the RNase is a non-specific RNase.
The invention also features a composition comprising a host cell that produces a recombinant DNA, a recombinant protein, or a recombinant carbohydrate and also produces an RNase in a regulated manner.
RNase produced in said regulated manner is overproduced, or inducibly overproduced, or constitutively overproduced. The RNase produced by the host cell also may be secreted out of the host cell cytoplasm, for example, secreted into the host cell periplasm or secreted out of the host cell into the medium.
In some methods, it is preferred that the RNase is a non-specific RNase.
The invention also features a composition comprising a host cell that produces a recombinant DNA, a recombinant protein, or a recombinant carbohydrate and a host cell that produces an RNase in a regulated manner.
RNase produced in said regulated manner is overproduced, or inducibly overproduced, or constitutively overproduced. The RNase produced by the host cell also may be secreted out of the host cell cytoplasm, for example, secreted into the host cell periplasm or secreted out of the host cell into the medium.
The invention also features a pharmaceutical composition comprising a cellular component that is substantially RNA-free, in a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition comprising a cellular component that is substantially RNA-free obtainable by the methods of the present invention, in a pharmaceutically acceptable carrier.
Preferably, the cellular component that is substantially RNA-free is made according the methods described herein.
As used herein, xe2x80x9csubstantially RNA-freexe2x80x9d and xe2x80x9csubstantially all of the RNA moleculesxe2x80x9d refers to the exceedingly small amount of RNA that is permitted in a preparation of a cellular component that is useful for administration to humans. Acceptable exceedingly low levels of RNA are as follows.
xe2x80x9cSubstantially RNA-free DNAxe2x80x9d refers to the presence in a sample containing the cellular component of less than 1%, preferably less than 0.2% and most preferably less than 0.1%-0.01% (w/w of RNA/DNA in the sample).
xe2x80x9cSubstantially RNA-free proteinxe2x80x9d refers to a protein preparation that contains less than 1% and preferably less than 0.2% and most preferably less than 0.1%-0.01% (w/w) of RNA/protein. When the protein is a therapeutic protein xe2x80x9csubstantially RNA-free proteinxe2x80x9d refers to a therapeutic protein preparation that contains less than 10 ng of RNA/dose, preferably less than 500 pg of RNA/dose, more preferably less than 100 pg of RNA/dose, most preferably less than 5-10 pg of RNA/dose.
xe2x80x9cSubstantially RNA-free carbohydratexe2x80x9d refers to a carbohydrate preparation that contains less than 1% and preferably less than 0.2% and most preferably less than 0.1%-0.01% (w/w) of RNA/carbohydrate.
xe2x80x9cCell lysatexe2x80x9d refers to the composition produced on lysing cells wherein the composition does not comprise exogenously produced RNase. Other exogenously produced components may be added to the cell lysate such as proteases or protease inhibitors.
Preferably, the cell lysate will contain significantly less RNase protein than prior art methods where exogenous RNase is added to cell lysate. xe2x80x9cSignificantly lessxe2x80x9d in this context refers to less than 10 xcexcg-50 xcexcg RNase/ml lysate, preferably less than 10 xcexcg RNase/ml of lysate and more preferably less than 1 xcexcg RNase/ml.
As used herein, xe2x80x9ccellular componentxe2x80x9d refers to any one of DNA, including but not limited to plasmid DNA, cosmid DNA, yac DNA, episomal DNA, or genomic DNA; recombinant protein, and recombinant carbohydrate.
As used herein, xe2x80x9crecombinantxe2x80x9d refers to DNA, protein or carbohydrate that is not naturally occurring in a cell or produced in an amount that is not normally produced by the cell.
As used herein, xe2x80x9ccellxe2x80x9d refers to any eukaryotic or prokaryotic cell. Preferred eukaryotic cells include HeLa cells, CHO cells, myeloma cell lines such as NSO, insect cells such as Sf21 and Sf9 cells, plant cells and lower eukaryotic cells including yeasts. Most preferably the term xe2x80x9ccellxe2x80x9d refers to a gram negative or gram positive bacteria, including but not limited to E. coli, Salmonella typhimurium, Bacillus spp., Streptomyces spp. and Pseudomonas aeruginosa. 
xe2x80x9cRegulated mannerxe2x80x9d refers to gene expression, for example, of an RNase gene in a host cell that is transcriptionally regulated, for example, constitutively or inducibly. It also refers to regulation at the level of protein production, for example, the use of a signal sequence or a fusion protein to direct a protein to the host cell periplasm or out of the host cell and into the medium surrounding the host cell.
xe2x80x9cInduciblexe2x80x9d or xe2x80x9cinductionxe2x80x9d refers to transcriptional activation or derepression; xe2x80x9cconstitutivexe2x80x9d refers to continuous transcription often at a constant level; xe2x80x9csignal sequencexe2x80x9d refers to an amino acid sequence that directs secretion of the protein into the periplasm or out of the cell; xe2x80x9csecretionxe2x80x9d refers to movement of a secreted protein out of the host cell cytoplasm and into the periplasm or host cell culture medium; xe2x80x9cperiplasmxe2x80x9d refers to the host cell compartment between inner and outer cell membranes of, for example, E. coli. 
xe2x80x9cOverexpressesxe2x80x9d or xe2x80x9coverproducesxe2x80x9d refers to gene expression at a level higher than that normally expressed in the host cell; xe2x80x9coverproducedxe2x80x9d refers to production in an amount higher than that normally produced by the cell. Thus, over-expression of a given gene or overproduction of a gene product or a recombinant DNA encompasses production of 10% or more, 50% or more, 100% or more, or even up to over 200-500% more of the gene product than the cell normally produces in the absence of the over-expressed gene.
The invention provides DNA, protein or carbohydrate preparations that are substantially free of contaminating RNA. The invention is particularly useful in providing preparations that involve administration to humans, but also for other uses, including for accurate determination of DNA concentrations by absorbance at 260 nm (where RNA also absorbs), and for investigating both DNA/RNA and DNA/protein interactions. Substantially RNA-free protein is required for therapeutic applications, for estimating protein concentrations by absorbance at 280 nm, for analysis of protein crystal structures, for studying protein/DNA and protein/RNA interactions, and for certain protein purification protocols where nucleic acid binding reduces the affinity of protein for ion-exchangers or reduces the capacity of ion-exchangers for protein.
The invention is applicable to any use requiring a preparation of a cellular component, such as DNA, protein, carbohydrate, that is substantially free of contaminating RNA, for example, for preparation of a cellular component such as DNA or a protein or a carbohydrate that is suitable for therapeutic use in humans.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.